The goal of this proposal is to use the voltage dependent anion channel (VDAC) found in the outer mitochondrial membrane as a model system in which to study the general phenomena of voltage regulation of ion channel function. It takes advantage of the uniqueness of VDAC as an experimental system in which to analyze voltage-sensitive ion channel function and of the elegance of molecular genetic analysis as is possible in yeast. VDAC has been isolated from the outer mitochondrial membrane of yeast and analyzed biochemically and biophysically (see Section II and accompanying manuscripts). Antibodies to this protein have been raised in rabbits, affinity purified and used to demonstrate antigenic conservation among VDACs from a wide range of organisms. The antibodies have been used as well to screen yeast genomic expression libraries. The expressed sequences in positive clones have then been used to isolate homologous sequences from both yeast genomic and cDNA libraries. Clones (cDNA) containing VDAC sequences have authenticated using hybrid select translation and immunoprecipitation. In addition, the amino acid sequence derived by DNA sequencing of cDNA clones aligns with the partial amino acid sequence of a CNBr fragment obtained from the purified protein. From the primary amino acid sequence, a tentative structural model of the VDAC protein can be constructed based on computer predictions of secondary structure. From these cloned genes, we will attempt to construct a system in which sufficient VDAC for biochemical and physiological analysis can be produced completely in vitro. The cloned genes will be manipulated in vitro, first by the introduction of random mutations to define domains of the protein involved in voltage-sensitivity followed by site-directed mutagenesis to investigate the role of specific or a series of specific amino acids. These mutant proteins, produced in vivo or in vitro, will then be physiologically examined in artificial planar bilayers.